This invention relates to delivery of small interfering RNAs (siRNAs) for disease treatment. More particularly, this invention relates to a hydrophobically modified oligoarginine for use in delivering siRNAs for treatment of a disease, such as cancer.
The phenomenon of RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) is an evolutionarily conserved biological response to double-stranded RNA (dsRNA) for degradation of the sequence-specific homologous mRNA. RNAi is initiated by the dsRNA-specific endonuclease, Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments between 21-23 nucleotides long, termed small interfering RNAs (siRNAs), which bind to the RNA-induced silencing complex (RISC) that recognizes target mRNA to be degraded. P. D. Zamore, T. Tuschl, P. A. Sharp & D. P. Bartel, RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals, 101 Cell 25-33 (2000); S. M. Elbashir, W. Lendeckel & T. Tuschl, RNA interference is mediated by 21- and 22-nucleotide RNAs, 15 Genes Dev. 188-200 (2001); J. B. Opalinska & A. M. Gewirtz, Nucleic-acid therapeutics: basic principles and recent applications, 1 Nat. Rev. Drug Discov. 503-514 (2002); S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber & T. Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, 411 Nature 494-498 (2001). Recently, it has become a useful tool for specific gene silencing and analysis of gene function.
To improve the efficiency of siRNA function, delivery systems need to be developed to effectively condense siRNA to small particles, thereby enhancing cellular uptake and protection from enzymatic degradation. Ideas based on polymeric delivery systems for plasmid DNA, J.-S. Kim, A. Maruyama, T. Akaike & S. W. Kim, In vitro gene expression on smooth muscle cells using a terplex delivery system, 47 J. Control. Rel. 51-59 (1997); S. Han, R. I. Mahato & S. W. Kim, Water-soluble lipopolymer for gene delivery, 12 Bioconjugate Chem. 337-345 (2001); D. Y. Furgeson, W. S. Chan, J. W. Yockman & S. W. Kim, Modified linear polyethylenimine-cholesterol conjugates for DNA complexation, 14 Bioconjugate Chem. 840-847 (2003); W. J. Kim et al., Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis, 106 J. Control. Rel. 224-234 (2005); J. H. Jeong et al., Anti-GAD antibody targeted non-viral gene delivery to islet beta cells, 107 J. Control. Rel. 562-570 (2005), have begun to facilitate development of siRNA delivery vehicles, J. C. Bologna, G. Dom, F. Natt & J. Weiler, Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides, 23 Nucleosides Nucleotides Nucleic Acids 1729-1731 (2003); B. Urban-Klein, S. Werth, S. Abuharbeid, F. Czubayko & A. Aigner, RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo, 12 Gene Ther. 461-466 (2005). Therefore, enhancement of transfection efficiency in vitro and in vivo has progressed rapidly. In fact, numerous attempts to enhance siRNA function have been made based on cationic lipopeptide, N. Unnamalai, B. G. Kang & W. S. Lee, Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells, 566 FEBS 307-310 (2004), steroid and lipid conjugates of siRNA, C. Lorenz, P. Hadwiger, M. John, H.-P. Vornlocher & C. Unverzagt, Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells, 14 Bioorg. Med. Chem. Lett. 4975-4977 (2004); S. Spagnou, A. D. Miller & M. Keller, Lipidic carriers of siRNA: Differences in the formulation, cellular uptake, and delivery with plasmid DNA, 43 Biochemistry 13348-13356 (2004), and block copolymer-coated calcium phosphate nanoparticles, Y. Kakizawa, S. Furukawa & K. Kataoka, Block copolymer-coated calcium phosphate nanoparticles sensing intracellular environment for oligodeoxynucleotide and siRNA delivery, 97 Control. Rel. 345-356 (2004). Recently, to enhance cellular uptake and transfection efficiency, arginine-rich cell membrane permeable carrier peptides, such as human immunodeficiency virus (HIV-1) TAT and antennapedia, have been developed as gene delivery vehicles. C. Rudolph et al., Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells, 278 J. Biol. Chem. 11411-11418 (2003); D. S. Manickam, H. S. Bisht, L. Wan, G. Mao & D. Oupicky, Influence of TAT-peptide polymerization on properties and transfection activity of TAT/DNA polyplexes, 102 J. Control. Rel. 293-306 (2005). In cell culture, a conjugate of an antisense oligonucleotide and the antennapedia peptide inhibited the translation of amyloid precursor protein at a concentration of 40 nM. B. Allinquant, P. Hantraye, P. Mailleux, K. Moya, C. Bouillot & A. Prochiantz, Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro, 128 J. Cell Biol. 919-927 (1995). In a separate study, the antennapedia-peptide-conjugated oligonucleotide against Cu/Zn superoxide dismutase showed a 100-fold higher efficiency in culture than did the oligonucleotide itself. C. M. Troy, D. Derossi, A. Prochiantz, L. A. Greene & M. L. Shelanski, L. Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway, 16 J. Neurosci. 253-261 (1996). Furthermore, arginine oligopeptides have been modified with several hydrophobic lipid molecules to enhance plasmid gene transfection. S. Futaki et al., Stearylated arginine-rich peptides: A new class of transfection systems, 12 Bioconjugate Chem. 1005-1011 (2001).
An effective water soluble lipopolymer carrier (WSLP) was made by combining the cationic headgroup of branched polyethylenimine (bPEI, MW=1.8 kDa) with a hydrophobic lipid anchor, cholesterol chloroformate. S. Han, R. I. Mahato & S. W. Kim, Water-soluble lipopolymer for gene delivery, 12 Bioconjugate Chem. 337-345 (2001). WSLP showed low cytotoxicity and enhanced transfection efficacy in vitro and in vivo. Id., J. W. Yockman, A. Maheshwari, S. Han & S. W. Kim, Tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p2CMVmIL-12 complexes, 87 J. Control. Rel. 177-186 (2003). The effectiveness of WSLP over bPEI was due to the modification of the inherent structural DNA complex to enhance the interaction with plasma membrane and facilitate endosomal escape.
While prior art carriers and methods of use thereof are known and are generally suitable for their limited purposes, they possess certain inherent deficiencies that detract from their overall utility. For example, carriers that provide enhanced cellular uptake, improved inhibition of nucleolytic inactivation, and low cost are needed.
In view of the foregoing, it will be appreciated that providing an improved polymeric carrier for use as a carrier in siRNA delivery would be a significant advancement in the art.